Mechanical loading of bone maintains bone mass and skeletal integrity. Loading stimulates new bone formation and unloading causes bone loss. Cyclooxygenase (COX)-2, the major enzyme for prostaglandin (PG) responses in bone, can enhance both bone formation and resorption and may mediate some effects of either loading or unloading. Our overall goal is to understand how mechanical signals are transmitted into biochemical signals and the role of COX-2 in these processes. Mechanical strains in bone can generate interstitial fluid flow that exerts fluid shear stress (FSS) on cells and induces COX-2. Because in vivo studies suggest that effects of short duration loading are not improved by longer duration, we believe the signaling cascade after removal of FSS will be important for determining outcomes of loading. In vitro, we will examine responses of COX-2, and selected other genes important for bone turnover, in osteoblastic and osteocytic cells during and after stopping FSS. We will determine if the protein kinase D (PKD) pathway mediates the FSS induction of COX-2 via activation of ERK after stopping FSS;if the transcription factors Cbfa1 (Runx2) and NFAT are mediators of the FSS induction of COX-2 transcription;and if the development of COX-2 expression after FSS also depends on regulation of mRNA stability. We will assess the role of COX-2 produced PGs, induced by FSS, in FSS effects on other genes using nonsteroidal anti-inflammatory drugs (NSAIDs) and cells derived from Cox-2+/+ and Cox-2-/- mice. In vivo, we will study effects of dynamic axial loading of the ulnae and effects of unloading of hind limbs by tail suspension. We will use mice with Cox-2 deletion conditionally targeted to osteoblastic and osteocytic cells with the 2.3-kb Co1a1 promoter driving Cre recombinase (Col1a1-Cre) or the 2.3-kb Col1a1 promoter driving Cre recombinase fused to a mutated ligand binding domain of the estrogen receptor (Col1a1-CreERT2), which becomes active only after administration of the synthetic estrogen antagonist 4-hydroxy-tamoxifen. We will examine potential compensation by cyclooxygenase (COX)-1 for absent COX-2 by comparing targeted Cox-2 deletion in Cox- 1+/+ and Cox-1-/- mice. We will measure bone gain or loss and gene expression at intervals after loading or unloading to determine effects of Cox-2 deletion (+/- Cox-1 deletion) on the response of other genes that may mediate effects of loading and unloading. Public health relevance: Physical activity that mechanically loads bone can increase bone mass and decrease bone loss. Osteoporosis, associated with bone loss and leading to debilitating skeletal fractures, increases with age and physical inactivity. A better understanding of how mechanical loading regulates bone may help us to develop better therapies for prevention and treatment of osteoporosis.